Recombinant human alpha-fetoprotein as a cell proliferative agent

ABSTRACT

A method of promoting bone marrow cell proliferation in a mammal by administering unglycosylated recombinant human alpha-fetoprotein is disclosed.

This is a continuation of application Ser. No. 08/377,316, filed Jan.24, 1995, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to cell growth and cell go culture.

Mammalian alpha-fetoprotein (AFP) is a 70,000-Da glycoprotein ofprimarily yolk sac and hepatic origin which is present in fetal blood inmilligram amounts during perinatal life. At birth serum AFP levels begina gradual decline to the low nanogram amounts normally found in theadult. Chemical analysis has shown that AFP molecules are composed ofsingle polypeptide chains containing about 4% carbohydrate.

SUMMARY OF THE INVENTION

I have discovered that unglycosylated recombinant humanalpha-fetoprotein made in a prokaryote (e.g., E. coli) is a cellproliferative agent, e.g., promotes the growth of bone marrow in vitro.

In general, the invention features a cell culture medium includingrecombinant human alpha-fetoprotein or a cell-stimulating fragment oranalog thereof. Preferably, such recombinant human alpha-fetoprotein isproduced in a prokaryotic cell (E. coli) and is unglycosylated.

Accordingly, the invention features a method of cell culture, saidmethod including (a) providing a cell culture medium includingrecombinant human alpha-fetoprotein; (b) providing a cell; (c) andgrowing the cell in the medium, where the cell proliferates, and ismaintained. Preferably, the cell is a mammalian cell. Examples of suchmammalian cells include bone marrow cells (e.g., a T cells, a naturalkiller cell, a lymphocyte, etc.), hybridomas or a genetically-engineeredcell line. Examples of other cells include hematopoietic cells such asstem cells, blast cells, progentior cells (e.g., erythroid progenitorcells such as burst-forming units and colony-forming units),myeloblasts, macrophages, monocytes, macrophages, lymphocytes,T-lymphocytes, B-lymphocytes, eosinophils, basophils, tissue mast cells,megarkaryocytes (see e.g., Best and Taylor's Physiological Basis ofMedical Practice, John B. West, ed., Williams & Wilkins, Baltimore). Inother preferred embodiments the method involves ex vivo cell culture.

In another aspect, the invention features a method for inhibitingmyelotoxcity in a mammal (e.g., a human patient) involving administeringto the mammal a therapeutically effective amount of recombinant humanalpha-fetoprotein or a myelotoxic-inhibiting analog or fragment thereof.Preferably, the recombinant human alpha-fetoprotein is produced in aprokaryotic cell (E. coli) and is unglycosylated.

In another aspect, the invention features a method of inhibitingsuppression of bone marrow cell proliferation in a mammal, the methodinvolving administering to the mammal an effective amount of recombinantalpha-fetoprotein or an anti-suppressive fragment or analog thereof.Preferably, the recombinant human alpha-fetoprotein is produced in aprokaryotic cell (e.g., E. coli) and is unglycosylated.

In another aspect, the invention features a method of promoting bonemarrow cell proliferation in a mammal, involving administering to themammal an effective amount of recombinant human alpha-fetoprotein or acell-stimulating fragment or analog thereof. Preferably, the recombinanthuman alpha-fetoprotein is produced in a prokaryotic cell (e.g., E.coli) and is unglycosylated.

In another aspect, the invention features a method of preventing bonemarrow cell transplantation rejection in a mammal, involvingadministering to the mammal an effective amount of recombinant humanalpha-fetoprotein or an anti-rejection fragment or analog thereof.Preferably, the recombinant human alpha-fetoprotein is produced in aprokaryotic cell (e.g., E. coli) and is unglycosylated.

By “cell-stimulating” is meant increasing cell proliferation, increasingcell division, promoting cell differentiation and/or development, orpromoting cell longevity.

By “therapeutically effective amount” is meant a dose of unglycosylatedrecombinant human alpha-fetoprotein or an cell-stimulating fragment oranalog thereof capable of stimulating the proliferation of a cell.

By “recombinant human alpha-fetoprotein” is meant a polypeptide havingsubstantially the same amino acid sequence as the protein encoded by thehuman alpha-fetoprotein gene (FIG. 1, SEQ ID NO:1) as described byMorinaga et al., Proc. Natl. Acad. Sci., USA 80: 4604 (1983). The methodof producing recombinant human alpha-fetoprotein in a prokaryotic cellis described in U.S. Pat. No. 5,384,250.

By “myelotoxic-inhibiting” is meant inhibiting myeloablation.

According to the invention, administration of recombinant humanalpha-fetoprotein (“rHuAFP”) (or a fragment or analog thereof) can be aneffective means for promoting and boosting cell growth in vitro, exvivo, or in vivo. Administration of such rHuAFP can also be an effectivemeans of preventing or treating or ameliorating myleotoxcemia in amammal.

The use of rHuAFP is especially advantageous since there are no knownadverse side effects related to human alpha-fetoprotein and it isbelieved that relatively high doses can be safely administered.Furthermore, the use of rHuAFP as a principal component of tissueculture media is advantageous since there is little potential forcontamination with pathogens.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

The drawing will first be described.

DRAWINGS

FIG. 1 is the nucleotide sequence (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of the cDNA encoding human alpha-fetoprotein.

FIG. 2 is the SDS-PAGE analysis of rHuAFP Fragment I (SEQ ID NO:3) (LaneA, MW marker; Lane B, natural human alpha-fetoprotein (AFP); Lane C,unpurified rHuAFP; Lane D, rHuAFP Fragment I, and Lane E, rHuAFP (aminoacids 1-590 of FIG. 1, SEQ ID NO:2).

FIG. 3 is a bar graph showing murine bone marrow proliferation inserum-free RPMI medium in the presence or absence of both 400 μg/mlrHuAFP and 5 μg/ml transferrin.

PRODUCTION OF RECOMBINANT HUMAN ALPHA-FETOPROTEIN

As summarized above, the invention includes therapies for promoting cellproliferation and for the prevention and treatment of a myelotoxiccondition involving administering recombinant human alpha-fetoprotein(“rHuAFP”) or fragments or analogs thereof. Methods for producing suchrHuAFP in a prokaryotic cell are described in U.S. Pat. No. 5,384,250.Methods for producing rHuAFP fragments and analogs will now be discussedin greater detail.

Fragments and Analogs

The invention includes biologically active fragments of rHuAFP. Abiologically active fragment of rHuAFP is one that possesses at leastone of the following activities: (a) directs a specific interaction witha target cell, e.g., binds to a cell expressing a receptor which isrecognized by rHuAFP (e.g., the membrane of a bone marrow cell); or (b)stimulates, increases, expands or otherwise causes the proliferation ofa cell (e.g., binds to a cell surface receptor to impart anproliferative or stimulating-signal). The ability of rHuAFP fragments oranalogs to bind to a receptor which is recognized by rHuAFP can betested using any standard binding assay known in the art. Biologicalactivity of a rHuAFP fragment or analog can also be tested according tostandard methods, e.g., those described herein.

In general, fragments of rHuAFP are produced according to the techniquesof polypeptide expression and purification described in U.S. Pat. No.5,384,250. For example, suitable fragments of rHuAFP can be produced bytransformation of a suitable host bacterial cell with part of anHuAFP-encoding cDNA fragment (e.g., the cDNA described above) in asuitable expression vehicle. Alternatively, such fragments can begenerated by standard techniques of PCR and cloned into the expressionvectors (supra). Accordingly, once a fragment of rHuAFP is expressed, itmay be isolated by various chromatographic and/or immunological methodsknown in the art. Lysis and fractionation of rHuAFP-containing cellsprior to affinity chromatography may be performed by standard methods.Once isolated, the recombinant protein can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology,Work and Burdon, eds., Elsevier, 1980).

A rHuAFP fragment may also be expressed as a fusion protein with maltosebinding protein produced in E. coli. Using the maltose binding proteinfusion and purification system (New England Biolabs), the cloned humancDNA sequence can be inserted downstream and in frame of the geneencoding maltose binding protein (malE), and the malE fusion protein canthen be overexpressed. In the absence of convenient restriction sites inthe human cDNA sequence, PCR can be used to introduce restriction sitescompatible with the vector at the 5′ and 3′ end of the cDNA fragment tofacilitate insertion of the cDNA fragment into the vector.

Following expression of the fusion protein, it can be purified byaffinity chromatography. For example, the fusion protein can be purifiedby virtue of the ability of the maltose binding protein portion of thefusion protein to bind to amylose immobilized on a column.

To facilitate protein purification, the pMalE plasmid contains a factorXa cleavage site upstream of the site into which the cDNA is insertedinto the vector. Thus, the fusion protein purified as described abovecan then be cleaved with factor Xa to separate the maltose bindingprotein from a fragment of the recombinant human cDNA gene product. Thecleavage products can be subjected to further chromatography to purifyrHuAFP from the maltose binding protein. Alternatively, a fragment ofrHuAFP may be expressed as a fusion protein containing a polyhistidinetag can be produced. Such an alpha-fetoprotein fusion protein may thenbe isolated by binding of the polyhistidine tag to an affinity columnhaving a nickel moiety which binds the polyhistidine region with highaffinity. The fusion protein may then be eluted by shifting the pHwithin the affinity column. The rHuAFP can be released from thepolyhistidine sequences present in the resultant fusion protein bycleavage of the fusion protein with specific proteases.

Recombinant HuAFP fragment expression products (e.g., produced by any ofthe prokaryotic systems described in U.S. Pat. No. 5,384,250 may beassayed by immunological procedures, such as Western blot,immunoprecipitation analysis of recombinant cell extracts, orimmunofluorescence (using, e.g., the methods described in Ausubel etal., Current Protocols In Molecular Biology, Greene PublishingAssociates and Wiley Interscience (John Wiley & Sons), New York, 1994).

Once a fragment of rHuAFP is expressed, it is isolated using the methodsdescribed supra. Once isolated, the fragment of rHuAFP can, if desired,be further purified by using the techniques described supra. Fragmentscan also be produced by chemical synthesis (e.g., by the methodsdescribed in Solid Phase Peptide Synthesis, 2nd ed., 1984, The PierceChemical Co., Rockford, Ill.).

The ability of a candidate rHuAFP fragment to exhibit a biologicalactivity of alpha-fetoprotein is assessed by methods known to thoseskilled in the art (e.g., those described herein).

The purified recombinant gene product or fragment thereof can then beused to raise polyclonal or monoclonal antibodies against the humanrecombinant alpha-fetoprotein using well-known methods (see Coligan etal., eds., Current Protocols in Immunology, 1992, Greene PublishingAssociates and Wiley-Interscience). To generate monoclonal antibodies, amouse can be immunized with the recombinant protein, andantibody-secreting B cells isolated and immortalized with anon-secretory myeloma cell fusion partner. Hybridomas are then screenedfor production of recombinant human alpha-fetoprotein (or a fragment oranalog thereof)-specific antibodies and cloned to obtain a homogenouscell population which produces monoclonal antibodies.

As used herein, the term “fragment,” as applied to a rHuAFP polypeptide,is preferably at least 20 contiguous amino acids, preferably at least 50contiguous amino acids, more preferably at least 100 contiguous aminoacids, and most preferably at least 200 to 400 or more contiguous aminoacids in length. Fragments of rHuAFP molecules can be generated bymethods known to those skilled in the art, e.g., proteolytic cleavage orexpression of recombinant peptides, or may result from normal proteinprocessing (e.g., removal of amino acids from nascent polypeptide thatare not required for biological activity).

Recombinant HuAFP fragments of interest include, but are not limited to,Domain I (amino acids 1 (Thr)-197 (Ser), see FIG. 1, SEQ ID NO:3),Domain II (amino acids 198 (Ser)-389 (Ser), see FIG. 1, SEQ ID NO:4),Domain III (amino acids 390 (Gln)-590 (Val), see FIG. 1, SEQ ID NO: 5),Domain I+II (amino acids 1 (Thr)-389 (Ser), see FIG. 1, SEQ ID NO:6),Domain II+III (amino acids 198 (Ser)-590 (Val), see FIG. 1, SEQ ID NO:7), and rHuAFP Fragment I (amino acids 266 (Met)-590 (Val), see FIG. 1,SEQ ID NO:8). Activity of a fragment is evaluated experimentally usingconventional techniques and assays, e.g., the assays described herein.

The invention further includes analogs of full-length rHuAFP orfragments thereof. Analogs can differ from rHuAFP by amino acid sequencedifferences, or by modifications (e.g., post-translationalmodifications) which do not affect sequence, or by both. Analogs of theinvention will generally exhibit at least 80%, more preferably 85%, andmost preferably 90% or even 99% amino acid identity with all or part ofa rHuAFP amino acid sequence. Modifications (which do not normally alterprimary sequence) include in vivo, or in vitro chemical derivatizationof polypeptides, e.g., acetylation, or carboxylation; such modificationsmay occur during polypeptide synthesis or processing or followingtreatment with isolated modifying enzymes. Analogs can also differ fromthe naturally occurring rHuAFP by alterations in primary sequence, forexample, substitution of one amino acid for another with similarcharacteristics (e.g., valine for glycine, arginine for lysine, etc.) orby one or more non-conservative amino acid substitutions, deletions, orinsertions which do not abolish the polypeptide's biological activity.These include genetic variants, both natural and induced (for example,resulting from random mutagenesis by irradiation or exposure toethanemethylsulfate or by site-specific mutagenesis as described inSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Press, 1989, or Ausubel et al., supra). Also included arecyclized peptide molecules and analogs which contain residues other thanL-amino acids, e.g., D-amino acids or non-naturally occurring orsynthetic amino acids, e.g., β or γ amino acids, or L-amino acids withnon-natural side chains (see e.g., Noren et al., Science 244:182, 1989).Methods for site-specific incorporation of non-natural amino acids intothe protein backbone of proteins is described, e.g., in Ellman et al.,Science 255:197, 1992. Also included are chemically synthesizedpolypeptides or peptides with modified peptide bonds (e.g., non-peptidebonds as described in U.S. Pat. Nos. 4,897,445 and 5,059,653) ormodified side chains to obtain the desired pharmaceutical properties asdescribed herein. Useful mutants and analogs are identified usingconventional methods, e.g., those described herein.

The cloning, expression, isolation and characterization of exemplaryrHuAFP fragments now follows. These examples are provided to illustrate,not limit, the invention.

Experimental

Materials and Methods

Polymerase Chain Reaction (PCR) rHuAFP Fragments

Plasmid constructs encoding fragments of human alpha-fetoprotein wereprepared using polymerase chain reaction (PCR) techniques known to thoseskilled in the art of molecular biology, using oligonucleotide primersdesigned to amplify specific portions of the human alpha-fetoproteingene (see e.g., PCR Technology, H. A. Erlich, ed., Stockton Press, NewYork, 1989; PCR Protocols: A Guide to Methods and Applications, M. A.Innis, David H. Gelfand, John J. Sninsky, and Thomas J. White, eds.,Academic Press, Inc., New York, 1990, and Ausubel et. al., supra).

The following six rHuAFP fragments were prepared to evaluate theirbiological activity (e.g., according to the methods disclosed herein):

Domain I Amino acids 1 (Thr)-197 (Ser), (FIG. 1, SEQ ID NO:3) Domain IIAmino acids 198 (Ser)-389 (Ser), (FIG. 1, SEQ ID NO:4) Domain III Aminoacids 390 (Gln)-590 (Val), (FIG. 1, SEQ ID NO:5) Domain I + II Aminoacids 1 (Thr)-389 (Ser), (FIG. 1, SEQ ID NO:6) Domain II + III Aminoacids 198 (Ser)-590 (Val), (FIG. 1, SEQ ID NO:7) rHuAFP Fragment I Aminoacids 266 (Net)-590 (Val), (FIG. 1, SEQ ID NO:8)Amino acid sequences were deduced from those shown for humanalpha-fetoprotein (1 (Thr)-590 (Val), SEQ ID NO:2) in FIG. 1. Fragmentsof rHuAFP designated Domain I, Domain II, Domain III, Domain I+II,Domain II+III and rHuAFP Fragment I were synthesized using standard PCRreaction conditions in 100 μL reactions containing 34 μL H₂O, 10 μL10×reaction buffer, 20 μL 1 mM dNTP, 2 μL DNA template (HuAFP cloned inpI18), appropriate 5′ and 3′ oligonucleotide primers (10 μL 10 pmol/μL5′ primer, 10 μL 10 pmol/μL 3′ primer), 1 μL glycerol, 10 μL DMSO, and 1μL Pfu polymerase (Stratagene, LaJolla, Calif.). Primers used for PCRamplifications were:

aDomI25 5′-AAAAAAGGTACCACACTGCATAGAAATGAA-3′ (SEQ ID NO:9) aDomI35′-AAAAAAGGATCCTTAGCTTTCTCTTAATTCTTT-3′ (SEQ ID NO:10) aDomII55-′AAAAAAATCGATATGAGCTTGTTAAATCAACAT-3′ (SEQ ID NO:11) aDomII35′-AAAAAAGGATCCTTAGCTCTCCTGGATGTATTT-3′ (SEQ ID NO:12) aDomII55′-AAAAAAATCGATATGCAAGCATTGGCAAAGCGA-3′ (SEQ ID NO:13) aDomIII35′-AAAAAAGGATCCTTAAACTCCCAAAGCAGCACG-3′ (SEQ ID NO:14) a5′rHuAFPFragment I 5′-AAAAAAATCGATATGTCCTACATATGTTCTCAA-3′ (SEQ ID NO:15)Accordingly, primer pairs DomI25 and DomI3, DomII5 and DomII3, DomIII5and DomIII3, 5′rHuAFP Fragment I and DomIII3, DomI25 and DomII3, andDomII5 and DomIII3 were used to isolate cDNA sequences of Domain I,Domain II, Domain III, rHuAFP Fragment I, Domain I+II, and DomainII+III, respectively, of rHuAFP. Annealing, extension, and denaturationtemperatures were 50° C., 72° C., and 94° C., respectively, for 30cycles. PCR products were purified according to standard methods.Purified PCR products encoding Domain I and Domain I+II were digestedindividually with KpnI and BamHI and cloned separately intoKpnI/BamHI-treated pTrp4. Purified PCR products encoding Domain II,Domain III, Domain II+III, and rHuAFP Fragment I were digestedindividually with Bsp106I and BamHI and were cloned separately intoBsp106I/BamHI-treated pTrp4. Each plasmid construct was subsequentlytransformed into competent E. coli cells. Since the expression productwill begin with the amino acid sequence encoded by the translation startsignal methionine, it is expected that such signal will be removed, orin any event, not affect the bioactivity of the ultimate expressionproduct.

Results

Expression and Purification

E. coli containing the expression plasmid encoding rHuAFP Fragment I wascultured and purified. FIG. 2 (lane D) shows the SDS-PAGE profile of thepurified rHuAFP Fragment I. N-terminal amino acid sequence analysisshowed that rHuAFP Fragment I possessed the amino acid sequenceSer₂₆₇-Tyr-Ile-Cys-Ser-Gln-Gln-Asp-Thr₂₇₅ (SEQ ID NO:16) whichcorresponds to the expected N-terminal amino acid sequence of rHuAFPFragment I (see FIG. 1, SEQ ID NO:8) where the initiating methionine iscleaved intracellularly.

Cell Culture Media

The invention provides a media containing rHuAFP (or a fragment oranalog thereof) for cell culture. While media of the invention generallydoes not require the use of serum (e.g., fetal bovine serum, calf serum,horse serum, normal mouse serum, human serum, porcine serum, rabbitserum etc.), since such rHuAFP is intended to replace or supplement theuse of serum, those skilled in the art will understand and recognizethat serum can be added if desired. Media formulations are generallyprepared according to methods known in the art. Accordingly, anystandard medium, e.g., RMPI-1630 Medium, CMRL Medium, Dulbecco'sModified Eagle Medium (D-MEM), Fischer's Medium, Iscove's ModifiedDulbecco's Medium, McCoy's Medium, Minimum Essential Medium, NCTCMedium, and the like can be formulated with rHuAFP (or a fragment oranalog thereof) at the desired effective concentration. If desired,media supplements, e.g., salt solutions (e.g., Hank's Balanced SaltSolution or Earle's Balanced Salt Solution), antibiotics, nucleic acids,amino acids, carbohydrates, and vitamins are added according to knownmethods. If desired, growth factors, colony-stimulating factors,cytokines and the like can also be added to media according to standardmethods. For example, media of the invention can contain any of thefollowing substances, alone or in combination, with rHuAFP (or afragment or analog thereof): erythropoietin, granulocyte/macrophagecolony-stimulating factor (GM-CSF), granulocyte colony-stimulatingfactor (G-CSF), macrophage colony-stimulating factor (M-CSF), aninterleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, etc.), insulin-growthfactor (IGF), transferrin, albumin, and stem-cell growth factor (SCF).Media of the invention are useful for culturing a variety of eukaryoticcells, e.g., mammalian cells, yeast cells, amphibian cells, and insectcells. Media can also be used for culturing any tissue or organ. Suchmedia can also be used in a variety of culture conditions and for avariety of biological applications. Examples of such culture conditionsinclude, without limitation, bioreactors (e.g., continuous or hollowfiber bioreactors), cell-suspension cultures, semisolid cultures, liquidcultures, and long-term cell suspension cultures. Media of the inventionare also useful for industrial applications, e.g., culturing hybridomacells, genetically-engineered mammalian cells, tissues or organs.

Recombinant Human Alpha-Fetoprotein as a Cell-Proliferative Agent

Cell growth-promoting attributes of rHuAFP (or a fragment or analogthereof) is evaluated by any standard assay for analysis of cellproliferation in vitro and in vivo. As discussed infra, the art providesanimal systems for in vivo testing of cell growth promoting or boostingcharacteristics of rHuAFP (or a fragment or analog thereof).Furthermore, a wide variety of in vitro systems are also available fortesting growth-promoting or growth-boosting aspects of rHuAFP (or afragment or analog thereof).

Any cell that proliferates in response to rHuAFP (or a fragment oranalog thereof) can be identified according to standard methods known inthe art. For example, proliferation of a cell (e.g., a bone marrow cell)can be monitored by culturing in a liquid media containing the testcompound, either alone or in combination with other growth factors,added artificially to a serum-free or serum-based medium. Alternatively,such bone marrow cells can be cultured in a semisolid matrix of diluteagar or methylcellulose, and the test compound, alone or in combinationwith other growth factors, can be added artificially to a serum-free orserum-reduced medium. In the semisolid matrix the progeny of an isolatedprecursor cell, proliferating in response to rHuAFP or a fragment oranalog thereof, remain together as a distinguishable colony. Forexample, a bone marrow cell may be seen to give rise to a clone of aplurality of bone marrow cells, e.g., NK cells. Such culture systemsprovide a facile way for assaying whether a cell responds to rHuAFP (afragment or analog thereof) either alone or in combination with othergrowth factors.

If desired, identification and separation of expanded subpopulations ofcells is performed according to standard methods. For example, cells maybe analyzed by fluorescence-activated cell sorting (FACS). Thisprocedure generally involves labelling cells with antibodies coupled toa fluorescent dye and separating the labeled cells from the unlabelledcells in a FACS, e.g., FACScan (Becton Dickson). Thus virtually any cellcan be identified and separated, e.g., by analyzing the presence of cellsurface antigens (see e.g., Shah et al., J. Immunol. 140:1861, 1988).When a population of cells is obtained, it is then analyzedbiochemically or, alternatively, provides a starting population foradditional cell culture, allowing the action of the cells to beevaluated under defined conditions in culture.

In one working example, the effect of rHuAFP (or a fragment or analogthereof) on the growth of human bone marrow cells is examined asfollows. In general, human bone marrow samples are obtained according tostandard procedures after informed consent. For example, bone marrow isobtained from the iliac crest of a healthy donor and the marrow cellsare diluted in phosphate-buffered saline at room temperature. Cells arethen washed and cultured in an appropriate growth medium. For example,cultures can be set up by inoculating bone marrow cells in 20-30 ml ofMcCoy's medium containing 50 U/ml penicillin, 50 U/ml streptomycin and 2mM L-glutamine. Cultures are incubated in the presence or absence of thetest compound alone, or in combination with other growth factors, e.g.,transferrin or GM-CSF. The cultures are subsequently incubated at 37° C.in a humidified atmosphere containing 5% CO₂, 5% O₂, and 90% N₂ for thedesired time period. Cell proliferation assays are performed accordingto standard methods. For example, replicate samples cultured in thepresence and absence of the test compound are analyzed by pulsing thecells with 1-2 μCi of ³HTdR. After an incubation period, cultures areharvested onto glass-fiber filters and the incorporated ³H measured byliquid scintillation. Comparative studies between treated and controlcells, e.g., cell cultured in the presence of rHuAFP versus cellscultured in the absence of rHuAFP, are used to determine the relativeefficacy of the test molecule in stimulating cell proliferation. Amolecule which stimulates cell proliferation is considered useful in theinvention.

To evaluate the proliferative effects of rHuAFP (or a fragment or analogthereof) e.g., the effect of the test compound on hematopoiesis in vivo,the test molecule is administered to sublethally irradiated mice (ormice treated with an immunosuppressive agent such as cyclosporine orFK-506, or a chemotherapeutic agent such as 5-fluorouracil orcyclophosphamide or any other method known in the art to deplete bonemarrow) and normal mice according to standard methods, e.g.,intravenously or intraperitoneally, at an appropriate dosage on a dailybasis. Generally, administration of the test compound to treated mice isinitiated prior to and/or after treating the animal, e.g., withsublethal radiation or immunotherapy or chemotherapy. Control animalsreceive a placebo, e.g., human serum albumin or diluent, similarlyadministered as for rHuAFP or related molecules. The effect of the testmolecule on hematopoiesis is monitored by standard techniques. Forexample, white blood cell count in peripheral blood and spleen in bothtreated and control animals are analyzed. Qualitative and quantitativeanalyses of bone marrow, e.g., lymphocytic lineage or myeloid lineage orany other cell type, can also be determined and analyzed according toconventional methods. Comparative data between treated and controlanimals are used to determine the relative efficacy of the test moleculein promoting cell proliferation, e.g., stimulates bone marrow cellproduction, mature B lymphocyte, thymocyte, or peripheral T lymphocytecell production. A test molecule which stimulates cell proliferation isconsidered useful in the invention.

The following example demonstrates that unglycosylated rHuAFP stimulatesthe growth of bone marrow cells in vitro. This example is provided toillustrate, not limit, the invention.

Experimental

Materials and Methods

Animals

Adult male and female CBA/J mice were obtained from the JacksonLaboratory (Bar Harbor, Me.). All mice were bred and maintained in ouranimal facility. Animals used in this study were 12 to 20 weeks old.

Cultures

Bone marrow cells were collected by flushing the tibias and femurs ofCBA/J mice with modified Dulbecco's phosphate-buffered saline (PBS)using a sterile syringe and 25-gauge needle. Homogenous single-cellsuspensions were obtained by the repeated passage of cell mixturesthrough a Pasteur pipet. All cells were washed twice by centrifugationat 250 g for 10 min in PBS and then assessed for viability by trypanblue dye exclusion. A cell viability of 95% or better was recorded inall experiments. Cells were then adjusted to the desired concentrationprior to use. Bone marrow cells (250,000) were cultured in 96-wellround-bottom microtiter plates (Flow Laboratories, Mississauga, Ontario,Canada). The culture medium was serum-free RPMI plus 4 mM L-glutamine,20 mM Hepes, 100 U/ml penicillin, 100 μg/ml streptomycin (GIBCOLaboratories, Burlington, Ontario, Canada), 5 μg/ml transferrin, and5×10⁻⁵ 2-mercaptoethanol (Eastman Chemicals Co., Rochester N.Y.). Cellswere cultured in the presence or absence of rHuAFP at a concentration of400 μg/ml, respectively. Total volume of all cultures was 0.2 ml.Cultures were maintained at 37° C. in 95% humidified air and 5% CO₂. Sixhours prior to harvesting, the cultures were pulsed with 1 μCi tritiatedthymidine (NEN, sp act 77.1 Ci/mmol). Cells were then harvested on glassfiber mats (Flow Labs) with a multiple sample harvester (Skatron, FlowLabs). Water-insoluble tritiated thymidine incorporation was measuredwith an LKB 1215 Rackbeta II using standard liquid scintillationtechniques.

rHuAFP

Recombinant HuAFP was synthesized and purified using the methodsdescribed in U.S. Pat. No. 5,384,250. Alternatively, rHuAFP can beobtained from Immtek, Inc. (Boston, Mass.).

Results

Effects of rHuAFP on Bone Marrow Proliferation in Serum-Free Media

The effects of purified rHuAFP on cultured murine bone marrow wasevaluated in serum-free medium. In this experiment, 2.5×10⁵ viable cellsfrom bone marrow of CBA/J mice were cultured for 72 hours in serum-freeRPMI media in the presence or absence of rHuAFP at a final concentrationof 400 μg/ml and transferrin at a final concentration of 5 μg/ml. Datashown in FIG. 3 indicate that bone marrow cells undergo a strongproliferative response in the presence of unglycosylated rHuAFP; with astimulation index (SI) of 35. No such proliferation was observed whenbone marrow cells were cultured in the absence of rHuAFP.

Therapy

As demonstrated above, rHuAFP is effective in promoting theproliferation of cells and accordingly is useful for therapy involvingthe promotion of cell proliferation, e.g., proliferation of bone marrowcells, and in treatment for the prevention of side effects ofimmunosuppressive therapy, radiotherapy or chemotherapy, or othertherapies known to depress the immune system and suppress bone marrowproduction, causing myelotoxicity. Accordingly, rHuAFP (or a fragment oranalog thereof) is employed to treat deficiencies in hematopoieticprogenitor or stem cells, or related disorders. Recombinant HuAFP (or afragment or analog thereof) may also be employed in methods for treatingcancer and other pathological states resulting in myelotoxcity, exposureto radiation or drugs, and including for example, leukopenia, bacterialand viral infections, anemia, B cell or T cell deficiencies, includingimmune cell or hematopoietic cell deficiency following autologous ornon-autologous bone marrow transplantation. Recombinant HuAFP (or afragment or analog thereof) may also be employed to stimulatedevelopment of megakaryocytes and natural killer cells in vitro or invivo.

The media, compositions, and methods of the invention are also usefulfor treating cancers that are treated by bone marrow transplants (BMT)that involve removing bone marrow cells from the patient, maintainingthese cells in an ex vivo culture while the patient is treated withradiation or chemotherapy, and then transplanting these cells back intothe patient after the treatment has been completed to restore thepatient's bone marrow. Accordingly, rHuAFP may be employed for BMT as ameans for reconstituting bone marrow in ex vivo cell culture medium andfor promoting bone marrow cell proliferation in vivo. Recombinant HuAFP(a fragment or analog thereof) is also useful for other cell therapies,e.g. cell expansion and/or gene therapy protocols, therapies requiringex vivo cell culture. Recombinant HuAFP (a fragment or analog) is alsouseful in the prevention of autologous or allogenic bone marrowtransplant rejection.

Therapeutic Administration

Recombinant HuAFP (or a fragment or analog thereof) can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions. Recombinant human alpha-fetoprotein, e.g., rHuAFP (or afragment or analog thereof), is preferably administered to the patientin an amount which is effective in preventing or ameliorating thesymptoms of myleotoxcity. Generally, a dosage of 0.1 ng/kg to 10 g/kgbody weight is adequate. For example, treatment of human patients willbe carried out using a therapeutically effective amount of rHuAFP (or afragment or analog thereof) in a physiologically acceptable carrier.Suitable carriers and their formulation are described for example inRemington's Pharmaceutical Sciences by E. W. Martin. The amount ofrHuAFP to be administered will vary depending upon the manner ofadministration, the age and body weight of the patient, and with thetype of disease, and size of the patient predisposed to or sufferingfrom the disease. Preferable routes of administration include, forexample, oral, subcutaneous, intravenous, intrperitoneally,intramuscular, transdermal or intradermal injections which providecontinuous, sustained levels of the drug in the patient. In otherpreferred routes of administration, rHuAFP can be given to a patient byinjection or implantation of a slow release preparation, for example, ina slowly dissociating polymeric or crystalline form; this sort ofsustained administration can follow an initial delivery of the drug bymore conventional routes (for example, those described above).Alternatively, rHuAFP can be administered using an external orimplantable infusion pump, thus allowing a precise degree of controlover the rate of drug release, or through installation of rHuAFP in thenasal passages in a similar fashion to that used to promote absorptionof insulin. As an alternative to nasal transmucosal absorption, rHuAFPcan be delivered by aerosol deposition of the powder or solution intothe lungs.

The therapeutic method(s) and compositions of the present invention mayalso include co-administration with other human growth factors.Exemplary cytokines or hematopoietins for such use include, withoutlimitation, factors such as an interleukin (e.g., IL-1), GM-CSF, G-CSF,M-CSF, tumor necrosis factor (TNF), transferrin, and erythropoietin.Growth factors like B cell growth factor, B cell differentiation factor,or eosinophil differentiation factors may also prove useful inco-administration with rHuAFP (or a fragment or analog thereof). Thedosage recited above would be adjusted to compensate for such additionalcomponents in the therapeutic composition. Progress of the treatedpatient can be monitored by conventional methods.

Treatment is started generally with the diagnosis or suspicion ofmyelotoxcity and is generally repeated on a regular or daily basis toameliorate or prevent the progression or exacerbation of the condition.Protection or prevention from the development of a myleotoxcemiccondition is also achieved by administration of rHuAFP prior to theonset of the disease. If desired, the efficacy of the treatment orprotection regimens is assessed with the methods of monitoring ordiagnosing patients for myelotoxcity.

The method(s) of the invention can also be used to treat non-humanmammals, for example, domestic pets, or livestock.

All publications, manufacturer's instructions, patents, and patentapplications mentioned in this specification are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

1. A method of promoting bone marrow cell proliferation in a mammal,said method comprising administering to said mammal an effective amountof recombinant human alpha-fetoprotein, wherein said recombinant humanalpha-fetoprotein is produced in a prokaryotic cell and isunglycosylated.
 2. The method of claim 1, wherein said prokaryotic cellis E. coli.